LABORATORY

WORK MANUAL

PHARMACOLOGY


PREPARED BY:
STAFF OF THE DEPARTMENT OF PHARMACOLOGY & THERAPY
FACULTY OF MEDICINE PUBLIC HEALTH AND NURSING
UNIVERSITAS GADJAH MADA
YOGYAKARTA

FOR:
FACULTY OF DENTISTRY
UNIVERSITAS GADJAH MADA
YOGYAKARTA

Laboratory Work Manual - Pharmacology l 1


























Laboratory Work Manual Pharmacology
ISBN N0 979-25-4437-2

Copyright@2016 by Faculty of Medicine, Gadjah Mada University

Printed in Yogyakarta

First printed: February 2009

2nd : March 2010
3rd : March 2011
4th : March 2012
5th : March 2013
6th : March 2014
7th : March 2015
8th : March 2016
9th: March 2017
10th:March 2019
11th :February 2020
12th : February 2021

Designed by: the Department of Pharmacology and Therapy Published by Faculty of Medicine,
Public Health, and Nursing Universitas Gadjah Mada All right reserved

This publication is protected by copyright law and permission should be obtained from publisher
prior to any prohibited reproduction, storage in a retrieval system, or transmission in any form
by any means, electronic, mechanical, photocopying and recording or likewise

2l Laboratory Work Manual - Pharmacology

 CONTRIBUTORS


Prof. dr. Ngatidjan, M.Sc., SpFK(K)
Dr. Med. dr. H. Widharto PH., SpFK.
Dra. Yuliastuti, Apt, M.Kes.
Dr. Maulina Diah S., Apt., M.Kes.
.Dr. Med. dr. Indwiani Astuti
Dr. dr. Setyo Purwono, M.Kes., SpPD.
Prof. dr. Iwan Dwiprahasto, M.Med.Sc., Ph.D.
Prof. Dr. Erna Kristin, MSi., Apt.
Prof. Dr. Mustofa, M.Kes., Apt.
Dr. dr. Eti Nurwening Sholikhah., M.Kes., M.Med.Ed
Dr. dr. Woro Rukmi Pratiwi, M.Kes, Sp.PD., FINASIM
dr. Jarrir At Thobari. Ph.D.
dr. Dwi Aris Agung Nugrahaningsih, M.Sc., Ph.D
drg. Mayu Winnie Rachmawati, M.Sc., Ph.D

Laboratory Work Manual - Pharmacology l

 P R E F A C E


The activity of laboratory work is one of the learning-teaching processes of Pharmacological
Science. Through this activity, students are able to learn about the pharmacological effects of
any medicines which have therapeutic meaning. Any medicine experiment tested on both
animals and human being is expected to easily help students understand the pharmacokinetic,
pharmacodynamical, and indication of medicine.
This laboratory work manual is shortly written and doesn’t explain the deep theory.
Furthermore, the students are strongly advised to firstly study the related text books or the
reference books, so that they are able to understand the basics and purpose of the experiment.
In practice, the results of the laboratory experiments among the groups are frequently
different depending on the techniques and the persons. Every experiment, even though repeated
several times, can show different results (there are various results) although they are in one range
For this reason, the results of all groups should be collected to be graded.
This Pharmacology laboratory work manual is aimed to help students to learn about
Pharmacology more systematically and comprehensively.

Thank you.

Yogyakarta, February 2021

Head of the Department of Pharmacology and Therapy
Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada

Dr. dr. Eti Nurwening S., M.Kes., M.Med.Ed

NIP. 196911021996012001

Coordinator Work Laboratory

Faculty of Dentistry, Universitas Gadjah Mada

drg. Mayu Winnie Rachmawati, M.Sc., Ph.D

NIP. 196911021996012001

4l Laboratory Work Manual - Pharmacology

 TABLE OF CONTENTS

Title ...................................................................................................................................... 1
Contributors ........................................................................................................................ 3
Preface ................................................................................................................................. 4
Table of Contents.................................................................................................................. 5
Rule and Regulation of Laboratory Work ............................................................................ 6
The Influence of Route of Drug Administration on Drug Onset and Duration ................... 10
Drug Excretion .............................................................................................................. 13
Dose-Effect Relationship .............................................................................................. 18
Analgesics .................................................................................................................................. 24
Local Anesthetics.......................................................................................................... 31
Acute Poisoning and Its Antidote .................................................................................. 34
References ................................................................................................................................. 37

Laboratory Work Manual - Pharmacology l

 RULE AND REGULATION OF ONLINE
LABORATORY WORK BY DEPARTMENT OF
PHARMACOLOGY AND THERAPY
FACULTY OF MEDICINE, PUBLIC HEALTH, AND NURSING
for
FACULTY OF DENTISTRY
UNIVERSITAS GADJAH MADA


1. The laboratory work begins on time as mentioned on the schedule.
2. The students should join on time. There will be a pre-test for 5 minutes before the laboratory
work begins
3. The students who join the link late for 5 minutes or more will not get a pre-rest mark, but the
student still allow to do the practical work.
4. The student who comes late for 15 minutes or more is not allowed to do the laboratory work
and must inhal next year.
5. The students should wear laboratory coat (snij jas) when enter the laboratory.
6. The students should wear dress properly
7. Whenever the students will do the laboratory work, the students should put the presence
by the link we shared.
8. At the laboratory work, each group will get a check list of laboratory equipment and materials
which will be used during the laboratory work.
9. Every student should do the assignment and discussion with the lecture systematically
based on the regulation after watch and observe the video in the breakroom.
10. The assignment must be submitted maximum in the same day after the each practical laboratory.
11. The student who is not able to attend to the inhal laboratory work on that day should follow
the practical laboratory on the next year.
12. The grade of the assignment is determined by the tidiness, systematic, and content
13. Total grade of the laboratory work consists of pre-test (20%) and assignment (30%), and
practical examination (50%)

6l Laboratory Work Manual - Pharmacology

 LABORATORY WORK MANUAL

Pharmacology
The influence of Route of Drug Administration on
Drug Onset and Duration
l
Drug Excretion
l
Dose-Effect Relationship
l
Analgesics
l
Local Anesthetics
l
Acute Poisoning and Its Antidote

Laboratory Work Manual - Pharmacology l

 The Influence of Route of Drug Administration on
Drug Onset and Duration

INTRODUCTION
As a rule, drugs reach their target organs via the blood. Therefore, they must first enter the
blood, usually the venous limb of the circulation. There are several possible sites of entry.
The drug may inject or infused intravenously, in which case the drug is introduced directly into
the bloodstream. I subcutaneous or intramuscular injection, the drug has to diffuse from its site
application in to blood. Because these procedures entail injury to the outer skin, strict requirements
must be met concerning technique. For that reason, the oral route involving subsequent uptake
of drug across the gastrointestinal mucosa into the blood is chosen much more frequently. The
disadvantage of this route is that the drug must pass through the liver on its way into the general
circulation. This fact assumes practical significance with any drug that may be rapidly transformed
or possibly inactivated in the liver (first pass hepatic elimination). Even with rectal administration,
at least a fraction of the drug enters the general circulation via the portal vein, because only vein
draining the sort terminal segment of the rectum communicate directly with the inferior vena
cava. Hepatic passage is circumvented when absorption occurs buccally or sublingually, because
venous blood from the oral cavity drains directly into the superior vena cava. The same would apply
to administration by inhalation. However, with this route, a local effect is usually intended; a
systemic action is intended only in exceptional cases. Under certain condition, drug can also
be applied percutaneously in the form of transdermal delivery system. In this case, drug is slowly
released from the reservoir, and then penetrates the epidermis and sub-epidermal connective tissue
where it enters blood capillaries. Only a very few drugs can be applied transdermally. The feasibility
of this route is determined by both the physicochemical properties of the drug and the therapeutic
requirements.
Speed of absorption is determined by the route and method of application. It is fastest with
intravenous injection, less fast which intramuscular injection, and slowest with subcutaneous
injection. When the drug is applied to the oral mucosa (buccal, sublingual route), plasma levels
rise faster than with conventional oral administration because the drug preparation is deposited
at its actual site of absorption and very high concentrations in saliva occur upon the dissolution of
single dose. Thus, uptake across the oral epithelium is accelerated. The same does not hold true
for poorly water-soluble or poorly absorbable drugs. Such agents should be given orally, because
both the volume of fluid for dissolution and the absorbing surface are much larger in the small
intestine than in the oral cavity.

EXPERIMENT
The aim of the experiment: To know and compare a drug effect in various route of
administration
a. Subject Rat
b. Equipment

8l Laboratory Work Manual - Pharmacology

 (1) 1 mL syringe
(2) Stomach tube
(3) Rat cage
(4) Rat fixators
c. Materials & Drugs
(1) Diazepam (0.5 mg/ml suspension, made from diazepam tablet that was diluted in sterile
water)
(2) Cotton
(3) Alcohol 70%
(4) Warm water
d. Procedure
(1) Students are divided into 5 groups.
(2) Each student groups works on three rats
l Rat A: will receive 4 ml (2 mg) diazepam orally
l Rat B: will receive 0.4 mL (2 mg) of diazepam intramuscularly
l Rat C: will receive 0.4 mL (2 mg) of diazepam intravenously
(3) Clean the area of injection by warm water and then by 70% alcohol (use cotton) before
injecting the drug.
(4) Observe and make record interval time between drug administration and sedation
(sedation onset of drug), between drug administration and sleep (sleep onset of drug),
sedation time, and sleeping time of each rat.
(5) Fall the mice from about 30 cm height. Normally, they will fall on their 4 feet. If not, they
show sedation effect. Sleeping time is time interval between fall in sleep and getting
up.

Table-1. Onset and duration of sedation effect of diazepam

Time
Route of drug Beginning of Lost of Onset Duration
Rat Administration Giving drug (minutes) (minutes)
effect effect
A Orally
B Intramuscularly
C Intravenously

Laboratory Work Manual - Pharmacology l

 Table-2. Onset and duration of sleeping effect of diazepam
Time
Route of drug Giving Beginning Loss of Onset Duration
Rat Administration drug of effect effect (minutes) (minutes)
A Orally
B Intramuscularly
C Intravenously

(6) Calculate the mean of onset and duration effect for each route of drug administration, and
compare the mean values for significance.

Table-3. Onset and duration of sedation effect of diazepam (all groups)

(Onset (minutes (Duration (minutes
Group Orally i.m .i.v Orally i.m .i.v
1
2
3
4
5
Mean
SD

Table-4. Onset and duration of sleeping effect of diazepam (all groups)

Onset Duration
Group
(minutes) (minutes)
Orally i.m i.v Orally i.m i.v
1
2
3
4
5
Mean
SD

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 Drug Excretion

INTRODUCTION
Most drugs are given orally and they must pass through the gut wall to enter the bloodstream.
This absorption process is affected by many factors such as:
a. Formulation
b. Stability to acid and enzyme
c. Motility of gut
d. Food in stomach
e. Degree of first pass metabolism
f. Lipid solubility (depends a lot on the pK of drug and pH environment)

But it is usually proportional to the lipid solubility of the drug. Thus, the absorption of unionized
molecules is favored because they are far more lipid soluble than those that are ionized and
surrounded by a shell of water molecules. Drugs are absorbed mainly from the small intestine
because of its large surface area. This is true even for weak acids (e.g., aspirin), which are non-
ionized in the acid (HCl) of the stomach. Drugs absorbed from the gastrointestinal tract enter the
portal circulation and some are extensively metabolized as they pass through the liver (first- pass
metabolism).

Route of administration
Drugs can be administered:
- Orally
- Intravenous injection
- Intramuscularly injection
- Subcutaneous injection
- Inhalation
- Topical
- Sublingual
- Rectal

Distribution
Distribution around the body occurs when the drug reaches the circulation. It must penetrate
tissues to act.
Drugs that are sufficiently lipid soluble to be readily absorbed orally are rapidly distributed
throughout the body water compartments. Many drugs are loosely bound to plasma albumin,
and an equilibrium forms between the bound and free drug in the plasma. Drug that is bound to
plasma proteins is confined to the vascular system and is not able to exert its pharmacological
actions.

Laboratory Work Manual - Pharmacology l

 If drug is given by intravenous injection, it enters the blood and is rapidly distributed to the
tissues. By taking repeated blood samples, the fall in plasma concentration of the drug with time
(i.e. the rate of drug elimination) can be measured. Often the concentration falls rapidly at first,
but then the rate of decline progressively decreases. Such a curve is called exponential, and this
means that at any given time a constant fraction of the drug present is eliminated in unit time.
Many drugs show an exponential fall in plasma concentration because the rates at which the drug
elimination processes work are themselves usually proportional to the concentration of drug in
the plasma.

The following processes are involved:

1. Elimination in the urine by glomerular filtration
2. Metabolism, usually by the liver
3. Uptake by the liver and subsequent elimination in the bile

A process that depends on the concentration at any given time is called first order and most
drugs exhibit first order elimination kinetics. If any enzyme system responsible for drug metabolism
becomes saturated, then the elimination kinetics change to zero order, i.e. the rate of elimination
proceeds at a constant rate and is unaffected by an increased concentration of the drug.

Excretion
Excretion, along with metabolism and tissue redistribution, is important in determining both
the duration of drug action and the rate of drug elimination. Excretion is a process whereby drugs
are transferred from the internal to the external environment, and the principal organs involved
in this activity are the kidneys, lungs, biliary system, and intestines.
Although some drugs are excreted through extra renal pathways, the kidney is the primary
organ of removal for most drugs, especially for those that are water-soluble and not volatile. The
three principal processes that determine the urinary excretion of a drug are glomerular filtration,
tubular secretion, and tubular reabsorption (mostly passive back-diffusion).
Excretion of drugs into sweat and saliva occurs but has only minor importance for most drugs.
The mechanisms involved in drug excretion are similar for sweat and saliva. Excretion mainly
depends on the diffusion of the un-ionized lipid-soluble form of the drug across the epithelial cells of
the glands.Thus, the pKa of the drug and the pH of the individual secretion formed in the glands are
important determinants of the total quantity of drug appearing in the particular body fluid. It is not
definitely established whether active drug transport occurs across the ducts of the glands.
Lipid-insoluble compounds, such as urea and glycerol, enter saliva and sweat at rates
proportional to their molecular weight, presumably because of filtration through the aqueous
channels in the secretory cell membrane. Drugs or their metabolites that are excreted into sweat
may be at least partially responsible for the dermatitis and other skin reactions caused by some
therapeutic agents. Substances excreted into saliva are usually swallowed, and therefore their
fate is the same as that of orally administered drugs (unless expectoration is a major characteristic of
a person’s habits). The excretion of a drug into saliva accounts for the drug taste patients
sometimes report after certain compounds are given intravenously.

12 l Laboratory Work Manual - Pharmacology

 Renal excretion is ultimately responsible for the elimination of most drugs. Drugs appear in the
glomerular filtrate, but if they are lipid soluble they are readily reabsorbed in the renal tubules by
passive diffusion. Metabolism of drug often results in a less lipid-soluble compound, aiding renal
excretion.
The ionization of weak acids and bases depends on the pH of the tubular fluid. Manipulation of
the urine pH is sometimes useful in increasing renal excretion.
Some drugs are concentrated in the bile and excreted into the intestine where they maybe
reabsorbed. This enterohepatic circulation increases the persistence of a drug in the body.

EXPERIMENT
Aim of experiment
The aim of experiment is to understand what happens to drugs after entering body.

Subject
Healthy volunteers, male, age between 20-40 years who have given informed consent will
participate in this experiment. All subjects should be in good health as assessed by medical
history and physical examination with no history of liver, kidney, or stomach disease and no history
of drug allergy.
Each subject receives oral dose of KJ (0,3g) with 200 mL of water.

Equipment
a. Test tubes and rack for test tubes
b. Pasteur pipettes
c. Measuring pipettes
d. Beaker glass

Material and Drugs

a. 0,3 g KI (in capsule)
b. Solution of 1% Kl
c. Solution of 10% NaNO2
d. Solution of dilute H2SO4
e. Solution of 1% amylum

Procedure
a. Each student group works with one volunteer.
b. Explain the procedure of the experiment to the volunteer.
c. Ask the volunteers to fill and sign the inform consent form once they understand about the
procedure of the experiment and agree to be the subject of the experiment.
d. Do anamnesis and vital sign examination to make sure those volunteers are healthy and
have no experience of allergic reaction to study drugs

Laboratory Work Manual - Pharmacology l

e. Ask the volunteer to emptying their bladder before taking medicine (Kl)
f. Take 2 mL of urine and 2 mL of saliva before the volunteer take medicine for control.
g. Give 0,3 g Kl to the volunteers and ask them to take it orally with 200mL of water.
h. Collect saliva sample every five minutes after drug administration.
i. Collect urine sample every fifteen minutes after drug administration.
j. Collect the sample until 90 minutes after drug administration.
k. Measure concentration of iod in urine and saliva by semi quantitative colorimetric. l. Do the
following reactions:
- Add 1 mL of 1% Kl in 1 % amylum, and monitor the color changing.
- One mL of 1% Kl + 2-3 drops of 10% NaNO2 + 2-3 drops of dilute H2SO4 + 1 drop of 1%
amylum (monitor the coloring changing)
- One mL of urine + 2-3 drops of 10% NaNO2 + 2-3 drops of dilute H2SO4 + 1 drop of 1%
amylum (monitor the color changing)
- One mL of saliva + 2-3 drops of 10% NaNO2 + 2-3 drops of dilute H2SO4 + 1 drop of 1%
amylum (monitor the color changing) Concentration of Iod is shown by color changing to
blue.
m. Semi quantitative of data is expressed as:
- negative (-)
- positive one (+)
- positive two (++)
- positive three (+++)
n. Semi quantitative data are tabulated and make curve of time vs. Iod concentration in urine
and saliva

14 l Laboratory Work Manual - Pharmacology

 Table-1. Iodium concentration in urine and saliva sample

Iodium concentration
Time saliva time urine
0’ 0’
5’
10’
15’ 15’
20’
25’
30’ 30’
35’
40’
45’ 45’
50’
55’
60’ 60’
65’
70’
75’ 75’
80’
85’
90’ 90’

Laboratory Work Manual - Pharmacology l

 Dose-Effect Relationship

INTRODUCTION
The effect of a substance depends on the amount administered, i.e., the dose. If the dose
choose is below the critical threshold (subliminal dosing), an effect will be absent. Depending on the
nature of the effect to be measured, ascending doses may cause the effect to increase in intensity.
Thus, the effect of an antipyretic or hypertensive drug can be quantified in a graded fashion, in
that the extent of fall in body temperature or blood pressure is being measured.
The dose-effect relationship may vary depending on the sensitivity of the individual person
receiving the drug, i.e., for the same effect, different doses may be required in different individuals.
Inter individual variation in sensitivity is especially obvious with effects of the all-or-none kind.
It could be illustrated by an experiment in which the subjects individually respond in all-or-
none fashion, as in the Straub tail phenomenon (Figure-A). Mice react to morphine with excitation,
evident in the form of an abnormal posture of the tail and limbs. The dose dependence of this
phenomenon is observed in groups of animals (e.g., 10 mice per group) injected with increasing
doses of morphine. At the low dose, only the most sensitive, at increasing doses a growing
proportion, at the highest dose all of the animals are affected (Figure-B).
There is a relationship between the frequency of responding animals and the dose given. At 2
mg/kg, one out of 10 animals reacts; at 10 mg/kg, 5 out of 10 respond. If the cumulative frequency
(total number of animals responding at a given dose) is plotted against the logarithm of the dose,
a sigmoid curve results (Figure-C). The curve point lies at the dose at which one-half of the group
has responded.The dose range encompassing the dose-frequency relationship reflects the variation
in individual sensitivity to the drug. Although similar in shape, a dose-frequency relationship has,
thus, a different meaning than does a dose-effect relationship. The latter can be evaluated in one
individual and results from an intra individual dependency of the effect on drug concentration.
The evaluation of a dose-effect relationship within a group of human subjects is compounded by
inter individual differences in sensitivity.To account for the biological variation, measurements have to
be carried out on a representative sample and the results averaged.Thus, recommended therapeutic
doses will be appropriate for the majority of patients, but not necessarily for each individual.The
variation in sensitivity may be based on pharmacokinetic differences (same dose different plasma
levels) or on differences in target organ sensitivity (same plasma level different effects).

Figure A. Abnormal posture in mouse given morphine

16 l Laboratory Work Manual - Pharmacology

Figure B. Incidence of effect as a function of dose

Figure C. Dose-frequency relationship

Laboratory Work Manual - Pharmacology l

Time Course of Plasma Concentration and Effect
After the administration of a drug, its concentration in plasma rises, reaches a peak, and then
declines gradually to the starting level, due to the processes of distribution and elimination.
Plasma concentration at a given point in time depends on the dose administered. Many drugs
exhibit a linear relationship between plasma concentration and dose within the therapeutic range
(dose-linear kinetics; (Figure-D); note different scales on ordinate). However, the same does
not apply to drugs whose elimination processes are already sufficiently activated at therapeutic
plasma levels so as to preclude further proportional increases in the rate of elimination when
the concentration is increased further. Under these conditions, a smaller proportion of the dose
administered is eliminated per unit of time.

Figure D. Dose linear kinetics

The time course of the effect and of the concentration in plasma is not identical, because the
concentration-effect relationship obeys a hyperbolic function (Figure-E). This means that the
time course of the effect exhibits dose dependence also in the presence of dose-linear kinetics
(Figure-F).

Figure E. Concentration Effect Relationship

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 Figure F. Dose dependence of the time course of the effect

In the lower dose range (example 1), the plasma level passes through a concentration range (0
→ 0.9) in which the concentration effect relationship is quasi-linear. The respective time courses
of plasma concentration and effect (D and F, left graphs) are very similar.
However, if a high dose (100) is applied, there is an extended period of time during which
the plasma level will remain in a concentration range (between 90 and 20) in which a change in
concentration does not cause a change in the size of the effect. Thus, at high doses (100), the time-
effect curve exhibits a kind of plateau. The effect declines only when the plasma level has returned
(below 20) into the range where a change in plasma level causes a change in the intensity of the
effect.
The dose dependence of the time course of the drug effect is exploited when the duration of
the effect is to be prolonged by administration of a dose in excess of that required for the effect.
This is done in the case of penicillin G as example, when a dosing interval of 8 hours is being
recommended, although the drug is eliminated with a half-life of 30 min. This procedure is, of
course, feasible only if supramaximal dosing is not associated with toxic effects.
Furthermore it follows that a nearly constant effect can be achieved, although the plasma
level may fluctuate greatly during the interval between doses. The hyperbolic relationship between
plasma concentration and effect explains why the time course of the effect, unlike that of the
plasma concentration, cannot be described in terms of a simple exponential function. A half-life
can be given for the processes of drug absorption and elimination, hence for the change in plasma
levels, but generally not for the onset or decline of the effect.
In this experiment, the dose-effect relationship is showed by increasing amount of saliva
after injecting increase dose of pilocarpine.

Laboratory Work Manual - Pharmacology l

EXPERIMENT
a. The aim of the experiment:
To know the dose-effect relationship

b. Subject:
Rat

c. Equipment:
(1) 1 mL syringe
(2) Rat cage

d. Materials & Drugs:

(1) Ketamine inj.
(2) Neostigmine
(3) Cotton
(4) 70% alcohol.

e. Procedure
(1) Each student groups works on 2 rats
(2) Clean the area of injection by 70% alcohol (use cotton) before injecting the drug.
(3) The rats were anesthetized with Ketamine (100mg/kg of body weight, ip)
(4) Before injecting physostigmine, insert pre-weighed absorbent foam cube sublingually for
10s. Weigh the foam. Amount of saliva produced was calculated as the change in the
weight of absorbent foam.
(5) Inject dose I of physostigmine intraperitoneally (5 μg).
(6) Ten minutes after injection, insert pre-weighed absorbent foam cube sublingually for
10s, and weigh it.
(7) Repeat procedure no 6 and 7 with dose II (5 μg) and dose III (10 μg) of physostigmine

20 l Laboratory Work Manual - Pharmacology

 Table-1. Amount of saliva produced (mg)

Amount of saliva (mg)

No of rat from each
group After injection of physostigmine at dose
Before injection of
physostigmine Dose I Dose II Dose III

1
Group 1
2

Group 2 3

Group 3 5

Group 4 7

Group 5 9

10

Mean ± SD

(8) Calculate the mean of amount of saliva for each collection and compare the mean
values for significance.

Laboratory Work Manual - Pharmacology l

 Analgesics

INTRODUCTION
Analgesic is a collective term of any member of the divers group of drugs used to relieve
pain. Analgesic drugs include the non-steroidal anti-inflammatory drugs (NSAIDs) (such as the
salicylates); narcotic drugs (such as codeine, morphine); and synthetic drugs with narcotic
properties (such as tramadol).
Narcotics analgesics exert their analgesic effect by stimulating endogenous opioid receptors.
Narcotic analgesics and the morphine-like synthetic drugs depress the central nervous system and
alter the perception of pain (Nociception). They are used to alleviate pain that cannot be relieved
by the NSAIDs. Most analgesics share common pharmacologic properties. These are: euphoria,
drowsiness, respiratory depression, nausea and vomiting, miosis, constipation, increase biliary
tract pressure, physiologic and psychological dependence, tolerance. Once patient develops
tolerance to one narcotic, they will show tolerance to another narcotics (cross tolerance).

Non-Opioid Analgesic
l Analgesic l Diclofenac potasium
l Aspirin l Ibuprofen
l Choline salicylate l Fenoprofen
l Magnesium salicylate l Ketoprofen
l Sodium salicylate l Naproxen
l Acetaminophen l Ketorolac
l Meclofenamate l Celecoxib
l Mefenamat acid

An NSAIDs are a group of compound with different chemical structure but having the same
therapeutics effect and side effect. They have anti-inflammatory, analgesic and antipyretic effect.
Their main mechanism of action is the inhibition of cyclooxygenase (COX) enzyme responsible
for prostaglandins (PGs) and thromboxane synthesis. Based on their structure, NSAIDs are
classified as: salicylic acid (aspirin), para-aminophenol (paracetamol), indole and indene acetic
acids (indomethacin, sulindac), hetero-aryl acetic acids (diclofenac, ketorolac), aryl-propionic
acids (ibuprofen, ketoprofen), anthranilic acids (mefenamic acid), enolic acids (piroxicam,
meloxicam), alkanones (nabumetone), pyrazolidinediones (phynylbutazone, oxyphenylbutazone,
diarylheterocycles (selective COX-2 inhibitors: celecoxib, rofecoxib). The inhibition of PGs leads to
the wide range of side effect, which includes gastrointestinal (GI) toxicity, cardiovascular events,
renal toxicity, exacerbation of hypertension and fluid retention. Non-selective NSAIDs lead to
greater risk of side effects, such as GI ulceration and bleeding.
Analgesics are frequently used in combination, such as paracetamol and ibuprofen
preparations found in many over the counter (OTC) pain relievers. They can also be found in
combination with vasoconstrictor drug such as Pseudoephedrine for sinus-related preparation, or
with anti-histamine drugs for allergy sufferers.

22 l Laboratory Work Manual - Pharmacology

IBUPROFEN
Ibuprofen is similar as an analgesic to aspirin, but not proven to have a clinically useful antiplatelet
activity. Ibuprofen has an approximately similar analgesic effect to paracetamol with addition of
anti-inflammatory effect.
Indication: pain and inflammation in rheumatic disease (including juvenile arthritis and other
musculoskeletal disorder; mild to moderate pain including dysmenorrhea; postoperative analgesia;
migraine; fever and pain in children.
Dose: initially 1,2-1,8 g daily in 3-4 divided doses preferably after food; increased if necessary to
max. 2,4 g daily; maintenance dose of 0,6-1,2 g daily may be adequate.
Juvenile rheumatoid arthritis, CHILD over 7 kg bodyweight 30-40 mg/kg daily in 3-4 divided doses.
Fever and pain in children, CHILD over 7 kg bodyweight 20-30 mg/kg daily in divided doses or 1-
2 years 50mg 3-4 times daily, 3-7 years 100mg 3-4 times daily, 8-12 years 200 mg 3-4 times daily.

PARACETAMOL (ACETAMINOPHEN)
Paracetamol is an antipyretic and analgesic with few, if any, anti-inflammatory properties.
Paracetamol inhibits prostaglandin biosynthesis under some circumstance (e.g. fever). Unlike
others NSAIDs, paracetamol does not have effect on platelet aggregation and gastric mucosa,
thereby can be use safely to aspirin intolerant patient. Moreover, it has not been associated with
Reye’s syndrome.
Indication: mild to moderate pain, pyrexia
Cautions: hepatic and renal impairment, alcohol dependence
Side effect: rare side-effect are rashes, blood disorder; important: liver damage (and less frequently
renal damage) following over dosage.
Dose: by mouth, 0,5-1 g every 4-6 hours to a max. of 4 g daily: CHILD 2 months 60 mg for post-
immunization pyrexia; otherwise under 3 months (on doctor’s advice only), 10 mg/kg (5 mg/kg
if jaundiced); 3 months-1 year 60-120 mg, 1-5 years 120-250 mg, 6-12 years 250-500 mg; these
doses may be repeated every 4-6 hours when necessary (max. of 4 doses in 24 hours).

EXPERIMENT
Aim of experiment
The aim of the experiment is to observe and to understand analgesic response after non-
narcotic analgesics administration.

Subjects
Fifteen healthy volunteers, male and female, age between 20-40 years who have given
informed consent will participate in this experiment. All subjects should be in good health as
assessed by medical history and physical examination. Each subject receives an oral dose of
ibuprofen (400 mg) or paracetamol (500mg) or placebo (glucose) with 200 mL of water.

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Equipment
a. Sphygmomanometer or blood pressure meter
b. Stopwatch
c. Stethoscope

Drugs requirement
a. Ibuprofen 400 mg
b. Paracetamol 500 mg
c. Glucose 500 mg

Procedure of experiment
a. Each student groups works with 3 volunteers.
b. Explain the procedure of the experiment to the volunteers. Written informed consent must be
signed by volunteers to show their agreement to be included in the experiment.
c. Make sure that volunteers are healthy and have no experiences of allergic reaction to study
drugs by doing anamnesis and vital sign examination.
d. Fix a manset of sphygmomanometer on left arm and pump the sphygmomanometer until 180
mmHg, hold the pressure on 180 mmHg for 3 minutes. Explain to the volunteers that the pain
that they feel at that time is 5 based on VAS pain quantification.
e. After above-mentioned procedure, give one of the drugs to the volunteers. Volunteers and
student are blind from the content of drug given.
f. Thirty minutes after ingestion of drugs or placebo, repeat and record the experiment procedure
(d).
g. Repeated experiments every twenty minutes.
h. Compare the observation result between pain sensation after and before drug or placebo
ingestion.
i. Make a graph (s) that showing the analgesics effect of drugs by time.

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 Informed Consent

Name : .........................................................................................................................

Age : .........................................................................................................................

Address : .........................................................................................................................

I herewith declare that I have received explanations and opportunities to ask something that I have
not understood about study of RESPONSE TO ANALGECIA AFTER ANALGESICS ADMINISTRATION
ON HEALTHY VOLUNTEERS. The explanations consist of the analgesics indication in the treatment,
the fate of analgesics in the body, the aim of experiment, the use of experiment, the procedure of
experiment and side effects that might occur during the experiment.

Procedures of experiment, which I agreed upon, are as follows:

1. Undergo clinical examinations by physician to determine my eligibility as a volunteer.
2. Answer questions for history taking, including history of allergy to drug, family health status,
meal habits etc.
3. Present in the laboratory as scheduled and undergo observation of pain sensation using
manset. Drug (placebo, 400 mg of ibuprofen or 500 mg of paracetamol) is taken with 200
ml of water. My blood pressure, heart rate, respiratory rate and subjective effects of pain
sensation will be measured serially, i.e before drug ingestion (time 0); and 30; 50; 70; and 90
minutes after drug or placebo ingestion.
4. Preventive measures have been prepared and all possible risk (if any), which may be caused
by the experiment, will be handled properly by supervisor.

I, therefore, agree to participate voluntarily in the experiment whithout ignoring my right to

withdrawn from the experiment at any time during my enrolment.

Yogyakarta, ………………….......….
.
Witnessed by: The undersigned,

(……………….........…………….) (……………………………………)
Student ID No..

Supervisor,

(………………………………………)

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 Table 1. 1 st volunteer

Name : ID No. 01
Age : Sex : Male / Female Weight (kg) : Height (cm) :
Adress :
History of:
1. Allergic reaction Yes / No
2. Asthma Yes / No
3. Anaphylactic shock Yes / No
Drug : Dosage : Oral
Respiratory rate : x / mnt Blood pressure : mmHg
Pulse rate : x / mnt

VAS
SCORE
Time
No. (minute) no pain severe pain
1 2 3 4 5 6 7 8 9 10
1. 0
2. 30
3. 60
4. 90
5. 120

Blood
Time Pulse rate Respiratory VAS Adverse
No. (minute) pressure
(x / mnt) rate (x / mnt) SCORE effect
(mmHg)

1.

2.

3.

4.

5.

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 Table . 2 nd volunteer

Name : ID No. 01

Age : Sex : Male / Female Weight (kg) : Height (cm) :

Adress :

History of:
1. Allergic reaction Yes / No
2. Asthma Yes / No
Drug : Dosage : Oral

Respiratory rate : x / mnt Blood pressure : mmHg

Pulse rate : x / mnt

VAS
SCORE
Time
No. (minute) no pain severe pain
1 2 3 4 5 6 7 8 9 10
1. 0
2. 30
3. 60
4. 90
5. 120

Blood
Time Pulse rate Respiratory VAS Adverse
No. (minute) pressure
(x / mnt) rate (x / mnt) SCORE effect
(mmHg)

1.

2.

3.

4.

5.

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 Table 3. 3 rd volunteer

Name : ID No. 01

Age : Sex : Male / Female Weight (kg) : Height (cm) :

Adress :

History of:
1. Allergic reaction Yes / No
2. Asthma Yes / No
3. Anaphylactic shock Yes / No

Drug : Dosage : Oral

Respiratory rate : x / mnt Blood pressure : mmHg

Pulse rate : x / mnt

VAS
S CORE
Time
No. (minute) no pain severe pain

1 2 3 4 5 6 7 8 9 10
1. 0
2. 30
3. 60
4. 90
5. 120

Time Blood Pulse Respiratory VAS

No. (minute pressure rate rate (x / SCORE Adverse effect
1.
2.
3.
4.
5.

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 Local Anesthetics

INTRODUCTION
Local anesthetics are drugs used to prevent pain by causing a reversible block of conduction
along nerve fibers. Most are weak bases that exist mainly in protonated form at body pH. The
drugs penetrate the nerve in a non-ionized (Lipophilic) form, but once inside the axon, some
ionized molecules are formed and these block the Na+ channels, preventing the generation of
action potentials.
All nerve fibers are sensitive to local anesthetics but, in general, small diameter fibers are
more sensitive than large fibers. Thus, a differential block can be achieved where the smaller
pain and autonomic fibers are blocked, whiles coarse touch and movement fibers are spared.
Local anesthetics vary widely in their potency, duration of action, toxicity and ability to penetrate
mucous membranes.
Local anesthetics depress other excitable tissue if the concentration in the blood is sufficiently
high, but their main systemic effects involve the central nervous system. Synthetic agents produce
sedation and light-headedness, although anxiety and restlessness sometimes occur, presumably
because central inhibitory synapses are depressed. Higher, toxic doses cause convulsions and
coma, with respiratory and cardiac depression, resulting from medullar depression.

Mechanism of Local Anesthetics

Local anesthetics penetrate into the interior of the axon in the form of lipid soluble free base.
There, protonated molecules are formed which then enter and block the Na+ channels after
binding to a receptor. Thus, quaternary (fully protonated) local anesthetics work only if they are
injected inside the nerve axon. Uncharged agents (e.g. benzocaine) dissolve in the membrane but
the channels are blocked in an all-or-none manner. Thus, ionized and non-ionized molecules act in
essentially the same way, that is, by binding to a receptor on the Na+ channel. Eventually so many
channels are inactivated that their number falls below the minimum necessary for depolarization
to reach threshold, and because action potentials cannot be generated, nerve block occurs.

Methods of administration
Surface anesthesia: Topical application to external or mucosal surfaces.
Infiltration anesthesia: Subcutaneous injection to act on local nerve endings, usually with a
vasoconstrictor.

Nerve block
Techniques range from infiltration of anesthetic around a single nerve (e.g. dental anesthesia)
to epidural and spinal anesthesia. In spinal anesthesia (intrathecal block) a drug is injected into the
cerebrospinal fluid in the subarachnoid space. In epidural anesthesia, the anesthetic is injected
outside the dura. Spinal anesthesia is technically far easier to produce than epidural anesthesia,
but the latter technique virtually eliminates the post-anesthetic complications such as headache

Laboratory Work Manual - Pharmacology l

Intravenous regional anesthesia. Anesthetic is injected intravenously into an exsanguinated limb.
A tourniquet prevents the agent reaching the systemic circulation.

EXPERIMENT
a. The purpose of the experiment:
(1) Understand effect of local anesthetic on pain stimulation
(2) Compare onset and duration between local anesthetic and saline solution
b. Subject:
Guinea-pigs
c. Equipment:
(1) Clippers
(2) Several 1 ml. Tuberculin syringes
(3) A stop watch or time clock (4) Darning needles
(5) Ballpoint
d. Materials & Drugs:
(1) Procaine hydrochloride 10 -2 M (or 3 mg. procaine hydrochloride/ml)
(2) NACL 0.9% 0.25 ml
The solutions should be made up in 0.9 per cent saline and be sterile.
The pH of the solution will affect the result and should be measured carefully and adjusted
to 7.4.if necessary
e. Procedure:
(1) Six Guinea-pigs are prepared at least 24 hours before the experiment by first clipping and
then shaving the hair on the lower back.
(2) Students are divided into 6 groups; each group of students works on 1 Guinea pig.
(3) The drug (0.25 ml) is injected intradermally (procaine hydrochloride 10 -2 M at left side
back and saline steril at right side back). Make outline in 3 cm diameter with a ball-point
(Fig. 1).
(4) Two minutes after the injection the sensitivity of the area is tested by pricking with a
needle, the skin at the site of injection and, as a control, the skin as far away from it as
possible.
(5) Twitches indicates no anesthesia (positive response), no twitches indicates anesthesia
(negative response). Records (+) for positive response and (-) for negative response.
(6) The test is repeated at 2- minute intervals, and records the response injection until show
positive response.

Minute 0 2 4 6 8 10 12 14 16 18 20 22 24 ...
P
L

P = Procaine hydrochloride 10 -2 M
L = NaCl 0.9% : 0.25ml

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(7) Record when the drug start acting (onset) and its duration (interval between the beginning
of negative response and positive response again).
(8) Record data from all groups into the table.

No. of Guinea Onset Duration

pig from each )minutes( )minutes(
group
P L C P L C

1
2
3
4
5
6

(9) Compare onset and duration of 2 kinds of local anesthetics by Student t-test.

Figure 1. Intradermally injection

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 Acute Poisoning and Its Antidote

INTRODUCTION
Poison is substance which in relatively small quantities is capable of producing a deleterious
response in a biological system or capable of destroying life. Toxicology is more than the science
of poisons. The notation includes its sources, its effect, its mechanism of action, the methods for
detecting poison including the diagnosis and therapy for poisoning.
Poisoning means an exposure of poison in the body producing damage response to health. The
toxic symptoms are related to the characteristics and conditions of exposure. These are related
to the dose delivered, form of substance, time and frequency of exposure and the route by which
exposure occurs.
Depending on the exposure time we’ll get an acute poisoning or the chronic one. It can be
strong, medium or weak toxicity. Other classification is to group them in categories that relate
to their source or to their usefulness. For example, we can group them to industrial toxic agents,
plant and animal materials, household mixtures and medicines. Another approach are based on
the organ or system that target site for the effect of the chemical (e.g. hepatotoxic, nephrotoxic,
neurotoxic) or the manner of exposure (e.g. inhalation toxicity). Whatever classification is selected,
it is inevitable that mixed classifications are found.
The most important aspect of the treatment of acute poisoning is supportive therapy to
maintain the vital signs (respiration and circulation). Another treatment is preventing of further
absorption of poison by using emetic, chemical absorption, purgative agent or gastric lavage.
Specific antidotes can detoxify poison by rendering it less toxic, by preventing its absorption, or by
enhance its biotransformation and its excretion. Excretion of a poison can be enhanced by forced
diuretics and by dialysis.
The first priority for inhalation, eyes and dermal exposure to poisons is to remove the patient
from the source of exposure. Eyes and skin should be thoroughly washed with large volume of
water. Emesis is contraindicated in certain situations: (1) if the patient is comatose or in state of
stupor or delirium; (2) if the patient has ingested a corrosive poison; (3) if the patient has ingested
a CNS stimulant; (4) if the patient has ingested petroleum distillate.
Antidotes can be grouped in categories that relate to their mechanism of action. They are
physical, chemical and physiological antidotes. Physical antidotes acts as an adsorbent and thereby
preventing absorption and poisoning (e.g. activated charcoal, milk flour, white of egg), chemical
antidotes bind the poison to prevent its absorption or to make it ineffective (e.g. poisoning of
alkaloids can be treated with KMnO4 and poisoning heavy metals can be treated with BAL).
Physiological antidotes give an opposite effect for poisoning effect (e.g. atropine is an antidote for
pilocarpine or other muscarinic agent).
In this experiment, we use cyanide as a strong toxic on for making a poisoning effect. Cyanide
can be found in household chemical properties, chemical industry or certain kind of tuberous
plant (e.g. potato, Manihot sp.), apple seed, peas and much other kind of plants. In a small dose,
ingested cyanide can be transformed by sulfur transferase enzyme (also called rhodanese) to
thiocyanate. Detoxification will be decreased if a gross of cyanide dose was ingested, producing a
potential poisoning. Cyanide reacts readily with the trivalent iron of cytochrome oxidize to form

32 l Laboratory Work Manual - Pharmacology

 the cytochrome oxidase-CN complex. Cytochrome oxidize has an important function in cellular
respiration and the loss of function will give a lack of oxygen symptom such as hypercapnea,
headache, tremor, palpitation, unconsciousness and asphyxia convulsions that may precede death.
Treatment for cyanide poisoning is specific and must be given rapidly if it is to prove effective.
The diagnosis can be made by the characteristic cyanide odor on the breath of the poisoned
individual. Cyanide poisoning can be treated by administration of nitrites and thiosulfate. The
nitrites change ferro (Hb) into methemoglobin (ferri-Hb).
Methemoglobin competes with cytochrome oxidize (Cyt-Fe+++) for the cyanide ion producing
cyanmethemoglobin and cytochrome oxidize is restored. Under the influence of sulphur
transferase, sodium thiosulfate reacts with cyanide to from thiocyanate, a relatively nontoxic
substance readily excreted in the urine.

EXPERIMENT
a. The Aim of the Experiment
To know the symptoms of poisoning and how to treat poisoning

b. Laboratory animal
Male and female guinea pig

c. Instruments
(1) Sterile injection and its needle
(2) Stethoscope, a pair of scale, a flash light
(3) Stopwatch

d. Material
(1) Alcohol 70 %
(2) KCN solution 0,25 %
(3) Na S O solution 10 %
(4) Cotton

e. Procedure
(1) Each student group works with a guinea pig
(2) Weigh the animal, notice and note its behavior (hyperactive, active, or hypoactive);
cyanosis (on ear, mouth and nose mucous); respiration (frequency, quality/type),
heartbeat; salivation; reflexes caused by exogen stimuli; tremor; convulsions.
(3) Inject KCN 5 mg/kg B.W. intraperitoneally (the dose should be correct). Note as for the
control every minute for about 5 minutes.
(4) When the poisoning symptoms appear clearly, inject Na-thiosulfate 250 mg/kg B.W.
intraperitoneally, and note the symptoms.
(5) Repeat the administration of Na-thiosulfate after 4 to 5 minutes the symptoms are still
there. Note until symptoms are vanished or the animal is dead.

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36 l

Table 1. Acute poisoning and Its Antidote

Before
After administration of
Observation Administration After administration of cyanide
Laboratory Work Manual - Pharmacology

Na-thiosulfate
of cyanide
5’ 10’ 15’ 20’ 25’ 30’ 5’ 10’ 15’ 20’ 25’ 30’
Behavior 1. hyperactive
2. active
3. hypoactive
Cyanosis Ear (+/-)
Mouth (+/-)
Nose (+/-)
Salivation (+/-)
Tremor (+/-)
Convulsion (+/-)
Eye reflexes Cornea (+/-)
Light (+/-)
Pupil size Cm
Respiration Frequency
(a/minute)
Type
1. abdominal
2. thoracal
Heartbeat Frequency
(a/minute)
REFERENCES
Goodman, AG, Goodman LS, Rall TW, Murrad F, 2007, Goodman and Gilman’s the Pharmacological
Basis of Therapeutics, Macmillan Publishing Company, New York, USA.
Lullman H, Mohr K, Ziegler A, Bieger D, 2000, Colour Atlas of Pharmacology, 2nd ed. Thieme
Stuttgart, New York, 2000
Vogel, HG (Ed), 2002, Drug Discovery and Evaluation Pharmacology Assays, 2nd ed., Springer-
Verlag, Berlin

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